One of the most visible signs of human aging are the changes experienced by the skin: dryness, appearance of spots, flaccidity and wrinkles. These effects can be caused by external agents such the constant exposure to the sun, atmospheric pollution or contact with chemical agents present in cleaning products for example, but are also the result of intrinsic physiological, biochemical and histological changes of the human organism, due to the decrease of the synthesis of proteins such as collagen or elastin, to an increase of proteolysis, and to a general breaking of the skin barrier, of the connective tissue and of cohesion.
Different active ingredients have been described for preventing and decreasing aging symptoms, such as retinoids, hydroxy acids, flavonoids or vitamin C and E derivatives, for example. Said compounds normally act by improving skin hydration, increasing cell renovation or preventing the degeneration of the tissue forming the skin; however, their efficacy in preventing and treating facial wrinkles caused by muscle contraction is limited. The facial expression wrinkle formation basis or mechanism is a tension of epidermal muscles dragging the skin inwardly. This muscular tension is the result of a hyperactivity of the nerves enervating facial muscles. Nervous hyperactivity is characterized by an uncontrolled and excessive release of neurotransmitters exciting muscular fibers. To that end, molecules regulating neuronal exocytosis contribute to relaxing muscular tension and subsequently, to eliminating facial wrinkles.
There is therefore a need to develop new active ingredients with proven efficacy for the preparation of a cosmetic or pharmaceutical composition for regulating neuronal exocytosis and, therefore for treating muscle spasticity and reducing and/or eliminating facial asymmetry and/or facial wrinkles, especially expression wrinkles.
Expression wrinkles are the wrinkles resulting from the stress exerted by the contractions of facial muscles responsible for causing facial expressions on the skin of the face. Expression wrinkles are usually located on the forehead, in the space between the eyebrows, around the mouth and/or around the eyes. Depending on the shape of the face, the expression frequency and the existence of tics (convulsive movements which are frequently repeated, caused by the involuntary contraction of one or several muscles, in this case facial muscles), expression wrinkles may even appear during adolescence. External factors such as exposure to the sun emphasize their depth and visibility.
Botulinum toxins have been widely used with the aim of reducing and/or eliminating expression wrinkles, especially serotype A (BOTOX® Cosmetic, Allergan Inc.) [Carruthers J. D. and Carruthers J. A. (1992) “Treatment of glabellar frown lines with C. botulinum-A exotoxin” J. Dermatol. Surg. Oncol. 18, 17-21; Mendez-Eastman S. K. (2003) “Botox: a review” Plast. Surg. Nurs. 23, 64-69]. The therapeutic and cosmetic treatment with BOTOX® consists of the localized injection of diluted pharmaceutical preparations (botulinum A-hemagglutinin complex, 500 kDa) in the areas in which the muscular tension is located. The paralytic effects of the toxin are reversible with an average duration of 6 months [Jankovic J. and Brin F. M. (1991) “Therapeutic uses of botulinum toxin” New Engl. J. Med. 324, 1186-1194; Jankovic J. (1994) “Botulinum toxin in movement disorders” Curr. Opin. Neurol. 6, 358-366]. The treatment therefore requires the repeared injection of botulinum toxin. The main problem of this treatment is the possibility of triggering an immune reaction against the pharmaceutical preparation due to the fact that its molecular size can be recognized by the patient's immune system. The appearance of antibodies against the botulinum toxin is a serious problem because it contributes to a clear loss of treatment efficacy [Jankovic J. and Brin F. M. (1991) “Therapeutic uses of botulinum toxin” New Engl. J. Med. 324, 1186-1194; Jankovic J. (1994) “Botulinum toxin in movement disorders” Curr. Opin. Neurol. 6, 358-366; Jankovic J. and Brin M. F. (1997) “Botulinum toxin: historical perspective and potential new indications” Muscle Nerve Suppl. 6, S129-5145; Davis L. E. (1993) “Botulinum toxin-from poison to medicine” West J. Med. 128, 25-28; Hughes A. J. (1994) “Botulinum toxin in clinical practise” Drugs 48, 888-893; Hambleton P. (1992) “Clostridium botulinum toxins a general review of involvement in disease, structure, mode of action and preparation for clinical use” J. Neurol. 239, 16-20; Borodic G. E. and Pearces L. B. (1994) “New concepts in botulinum toxin therapy” Drug Safety 11, 145-152; Brin M. F., Blitzer A., Stewart C., Pine Z., Borg-Stein J., Miller J., Nagalapura N. S. and Rosenfeld D. B. (1993) “Disorders with excessive muscle contraction: Candidates for treatment with intramuscular botulinum toxin (BoTox®)” Botulinum and Tetanus Neurotoxins (Ed. B. R. DasGupata), 559-576]. This loss of treatment efficacy with BOTOX® entails the need to increase the concentration of the preparation in subsequent treatments, which in turn causes a potentiation of the immune response. As an alternative to the treatment with botulinum toxin serotype A, the use of different serotypes of botulinum toxins, such as BoTox B, BoTox F and BoTox E, has been considered. Nevertheless, the application of pharmaceutical applications with different serotypes cannot be considered a solution to the problem, because sooner or later, the immune reaction can occur again. Furthermore, the treatment with botulinum toxins is expensive, mainly due to the lability and instability of the pharmaceutical preparations containing them.
There is therefore a pressing need to develop molecules imitating the paralytic effects of botulinum toxins but that are provided with much simpler and stabler molecular structures which do not induce immune reactions and the production cost of which is cost-effective. Molecules with a peptide nature comply with these properties.
At a molecular level, botulinum toxins are proteases degrading neuronal proteins that are involved in the calcium ion-activated exocytosis mechanism [Schiavo G., Rossetto O. and Montecucco C. (1996) “Bases Moleculares del tétanos y del botulismo” Investigación y Ciencia 234, 46-55; Montecucco C. and Schiavo G. (1994) “Mechanism of action of tetanus and botulinum neurotoxins” Mol. Microbiol. 13, 1-8; Schiavo G., Rosetto O., Benfenati F., Poulain B. and Montecucco, C. (1994) “Tetanus and botulinum neurotoxins are zinc proteases specific for components of the neuroexocytosis apparatus” Ann. NY Acad. Sci. 710, 65-75]. For example, botulinum toxin A, the most commonly used in clinical practice and cosmetics due to its applications in the elimination of facial wrinkles and facial asymmetry, and to ease the symptomatology of spasmodic diseases, truncates the neuronal SNAP-25 protein. This SNAP-25 protein has an important role in neurosecretion because it is involved in the formation of a protein complex (known as SNARE or fusion complex) directing and controlling the release of acetylcholine accumulated in vesicles. The core of said fusion complex is formed by syntaxin and SNAP-25 proteins, located in the presynaptic plasma membrane, and the synaptobrevin or VAMP protein, located in the vesicular plasma membrane [Calakos N. and Scheller R. H. (1996) “Synaptic vesicle biogenesis, docking and fusion: a molecular description” Physiol. Rev. 76, 1-29; Sutton R. B., Fasshauer D., Jahn R. and Brunger A. T. (1998) “Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4Å resolution” Nature 395, 347-3531 The main function of the fusion complex is to move the vesicle loaded with neurotransmitter (acetylcholine) closer to and place it in contact with the presynaptic plasma membrane [Calakos N. and Scheller R. H. (1996) “Synaptic vesicle biogenesis, docking and fusion: a molecular description” Physiol. Rev. 76, 1-29; Sutton R. B., Fasshauer D., Jahn R. and Brunger A. T. (1998) “Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4Å resolution” Nature 395, 347-353]. The fusion of both plasma membranes in response to a calcium concentration elevation is thus favored, thus causing the release of the neurotransmitter. Said vesicular fusion and anchoring SNARE protein complex therefore forms a key target for controlling neurosecretion. The truncation of any of the proteins forming the fusion complex prevents its assembly and therefore inhibits vesicular release and regulates neuronal exocytosis.
It is known in the state of the art that certain peptides derived from the sequences of the proteins forming the SNARE complex can inhibit neuronal exocytosis, such as for example peptides derived from the amino and carboxyl domains of the SNAP-25 protein [Apland J. P., Biser J. A., Adler M., Ferrer-Montiel A. V., Montal M., Canaves J. M. and Filbert, M. G. (1999) “Peptides that mimic the carboxy-terminal domain of SNAP-25 block acetylcholine release at an aplysia synapse” J. Appl. Toxicol. 19, Suppl. 1: S23-S26; Mehta P. P., Batternger E. and Wilson M. (1996) “SNAP-25 and synaptotagmin involvement in the final Ca2+-dependent triggering of neurotransmitter exocytosis” Proc. Natl. Acad. Sci. USA 93, 10471-10476; Ferrer-Montiel A. V., Gutierrez L. M., Apland J. P., Canaves J. M., Gil A., Viniegra S., Biser J. A., Adler M. and Montal M. (1998) “The 26-mer peptide released from cleavage by botulinum neurotoxin E inhibits vesicle docking” FEBS Lett. 435, 84-88; Gutierrez L. M., Canaves J. M., Ferrer-Montiel A. V., Reig J. A., Montal M. and Viniegra S. (1995) “A peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca2+-dependent exocytosis in chromaffin cells” FEBS Lett. 372, 39-43; Gutierrez L. M., Viniegra S., Rueda J., Ferrer-Montiel A. V., Canaves J. M. and Montal M. (1997) “A peptide that mimics the C-terminal sequence of SNAP-25 inhibits secretory vesicle docking in chromaffin cells” J. Biol. Chem. 272, 2634-2639; Blanes-Mira C, Valera E., Fernández-Ballester G., Merino J. M., Viniegra S., Gutierrez L. M., Perez-Payá E. and Ferrer-Montiel A. (2004) “Small peptides patterned after the N-terminus domain of SNAP-25 inhibit SNARE complex assembly and regulated exocytosis” J. Neurochem. 88, 124-135], the peptides derived from the amino acid sequence of syntaxin [Martin F., Salinas E., Vazquez J., Soria B. and Reig J. A. (1996) “Inhibition of insulin release by synthetic peptides show that the H3 region at the C-terminal domain of syntaxin-1 is crucial for Ca2+-but not for guanosine 5′-[gamma-thio]thriphosphate-induced secretion” Biochem. J. 320, 201-2051, of the sinaptobrevina [Cornille F., Deloye F., Fournie-Zaluski M. C., Rogues B. P. and Poulain B. (1995) “Inhibition of neurotransmitter release by synthetic proline-rich peptides shows that the N-terminal domain of vesicle-associated membrane protein/synaptobrevin is critical for neuro-exocytosis” J. Biol. Chem. 270, 16826-16830], of synaptotagmin [Mehta P. P., Batternger E. and Wilson M. (1996) “SNAP-25 and synaptotagmin involvement in the final Ca2+-dependent triggering of neurotransmitter exocytosis” Proc. Natl. Acad. Sci. USA 93, 10471-10476] and of the snapin protein [Ilardi J. M., Mochida S. and Sheng Z. H. (1999) “Snapin: A SNARE associated protein implicated in synaptic transmission” Nat. Neurosci. 2, 119-124]. In the same way, synthectic peptides obtained by rational design or by tracing synthetic chemical libraries which can interfere in the formation of the SNARE complex, inhibiting neuronal exocytosis, have also been described [Blanes-Mira C., Pastor M. T., Valera E., Fernández-Ballester G., Merino J. M., Gutierrez L. M., Perez-Paya E. and Ferrer-Montiel A. (2003) “Identification of SNARE complex modulators that inhibit exocytosis form an α-helix-constrained combinatorial library” Biochem J. 375, 159-166].
The industrial application of this type of compounds has been limited. The cosmetic industry has carried out considerable efforts to develop compounds imitating the action of botulinum toxins with exclusive use in treating and preventing the formation of expression wrinkles [Blanes-Mira C., Clemente J., Jodas G., Gil A., Fernández-Ballester G., Ponsati B., Gutierrez L. M., Pérez-Payá E. and Ferrer-Montiel, A. (2002) “A synthetic hexapeptide (Argireline®) with anti-wrinkle activity” Int. J. Cosmetic Res. 24, 303-310]. Specifically, patent EP1,180,524 of Lipotec, S. A. describes peptides derived from the amino-terminal fragment of the SNAP-25 protein having an anti-wrinkle effect, and international patent application WO97/34620 also describes peptides derived from the amino acid sequence of the SNAP-25 protein, specifically from its carboxy-terminal region, or from synaptobrevin or from syntaxin which can inhibit neuronal exocytosis.
None of the patents described above relates to irreversibly chemically modified derivatives of the SNAP-25 protein as regulating agents of neuronal exocytosis. Patent EP1,180,524 describes potential reversible chemical modifications of the peptides of the amino-terminal end of the SNAP-25 protein for the purpose of increasing its bioavailability and its ease in passing through the blood-brain barrier and epithelial tissue, such as the esterification of the side chains of aspartic and glutamic residues, which will subsequently be degraded in vivo by intracellular esterases, recovering the unmodified peptide responsible for biological activity. Surprisingly, the applicant of the present invention has discovered that chemically irreversible modifications of the amino and carboxyl ends of said peptides not only provide them with greater resistance to degradation against intracellular proteases, causing a longer duration of their effect as neuronal exocytosis regulators, but surprisingly, they can increase their in vitro efficacy from two to thirty times with respect to that of the corresponding unmodified peptide.
The modification of proteins with lipid chains is described as an irreversible modification when it is carried out on amino groups present in their sequences, either in their amino-terminal end or in side chains of lysine residues, whereas it is considered reversible when it is carried out on the thiol groups of cysteine residues, because the modified peptide or protein are hydrolyzed in vivo by the corresponding thioesterases [Magee A. I. (1990) “Lipid modification of proteins and its relevance to protein targeting” J. Cell Sci. 97, 581-584; Mumby S. M. (1997) “Reversible palmitoylation of signaling proteins” Curr. Opin. Cell Biol. 9, 148-154]. The state of the art describes examples of irreversible modifications of peptides with fatty acid chain derivatives for the purpose of improving their in vivo efficacy, increasing their permeation through the skin [Lintner K. and Peschard O. (2000) “Biologically active peptides: from a laboratory bench curiosity to a functional skin care product” Int. J. Cosmet. Sci. 22, 207-218] or achieving a better immunological response for their development as potential vaccines [Gahery H., Choppin J., Bourgault I., Fischer E., Maillere B. and Guillet J. G. (2005) “HIV preventive vaccine research at the ANRS: the lipopeptide vaccine approach” Therapie 60, 243-248], as well as for inducing a greater cytotoxic effect on bacteria [Eisenstein B. I. (2004) “Lipopeptides, focusing on daptomycin, for the treatment of Gram-positive infections” Expert Opin. Investig. Drugs 13, 1159-1169] or on fungi [Avrahami D. and Shai Y. (2004) “A New Group of Antifungal and Antibacterial Lipopeptides Derived from Non-membrane Active Peptides Conjugated to Palmitic Acid” J. Biol. Chem. 279, 12277-12285]. This type of modifications does not always cause a change in the in vitro efficacy of said peptides; for example the palmitoylation of the GHK tripeptide does not modify its capacity to induce collagen synthesis in fibroblasts [Lintner K. and Peschard O. (2000) “Biologically active peptides: from a laboratory bench curiosity to a functional skin care product” Int. J. Cosmet. Sci. 22, 207-218], so a person skilled in the art at the time of the present invention could not predict if the modification of a peptide with a hydrocarbon group would increase, decrease or leave its in vitro efficacy unaltered with respect to the corresponding unmodified peptide.
The irreversible chemical modification of peptides and proteins by means of the covalent incorporation of repetitive polyethylene glycol units, known as “PEGylation”, basically for the purpose of increasing the half-life period in vivo, decreasing the toxicity and reducing the imunogenicity and antigenicity of peptides and proteins, is also known in the state of the art [Savoca K. V., Abuchowski A., van Es T., Davis F. F. and Palczuk N. C. (1979) “Preparation of a non-immunogenic arginase by the covalent attachment of polyethylene glycol” Biochim. Biophys. Acta 578, 47-53; Hershfield M. S., Buckley R. H., Greenberg M. L., Melton A. L., Schiff R., Hatem C., Kurtzberg J., Marked M. L., Kobayashi R. H., Kobayashi A. L., et al. (1987) “Treatment of adenosine deaminase deficiency with polyethylene glycol-modified adenosine deaminase” N. Engl. J. Med. 316, 589-596; Katre N. V. (1990) “Immunogenicity of recombinant IL-2 modified by covalent attachment of polyethylene glycol” J. Immunol. 144, 209-213; Wang Q. C., Pai L. H., Debinski W., FitzGerald D. J. and Pastan I. (1993) “Polyethylene glycol-modified chimeric toxin composed of transforming growth factor oc and Pseudomonas exotoxin” Cancer Res. 53, 4588-4594; Clark R., Olson K., Fuh G., Marian M., Mortensen D., Teshima G., Chang S., Chu H., Mukku V., Canova-Davis E., Somers T., Cronin M., Winkler M. and Wells J. A. (1996) “Long-acting growth hormones produced by conjugation with polyethylene glycol” J. Biol. Chem. 271, 21969-21977; He X. H., Shaw P. C. and Tam S. C. (1999) “Reducing the immunogenicity and improving the in vivo activity of trichosanthin by site-directed pegylation” Life Sci. 65, 355-368; Harris J. M. and Chess R. B. (2003) “Effect of PEGylation on pharmaceuticals” Nat. Rev. Drug Discov. 2, 214-221]. The examples existing in the state of the art describes modifications of peptides and proteins for the purpose of improving their pharmacological properties of distribution and elimination and thus improving their in vivo biological activity without altering their in vitro biological activity, but in no case do they suggest that the potential PEGylation can increase the in vitro biological activity of the protein, rather on the contrary, there are described examples such as the case of PEGylated interferon in which the in vitro activity is reduced, comparing it with that of the native interferon [Rajender Reddy K., Modi M. W. and Pedder S. (1992) “Use of peginterferon alfa-2a (40 KD) (Pegasys) for the treatment of hepatitis C” Adv. Drug Deliv. Rev. 54(4), 571-86].
Surprisingly, the present invention shows that the irreversible chemical modification of peptide sequences derived from the SNAP-25 protein can increase the efficacy of said sequences in neuronal exocytosis inhibition. There is no indication in the state of the art that said modifications must increase the inhibitory effect of said peptides, therefore a person skilled in the art could not deduce the nature of the required modifications of the peptides to increase their capacity to inhibit neuronal exocytosis.
The present invention thus provides a novel solution to the existing needs, comprising the discovery of irreversibly chemically modified peptide sequences derived from the SNAP-25 protein that can inhibit neuronal exocytosis in a more effective and prolonged manner than the corresponding unmodified peptides that are already known in the state of the art.